Instant Volume Microscopy of Organoids with SOLIS

Advancements in microscopy techniques have revolutionized our ability to explore the intricacies of biological systems, with engineered human heart tissue (EHT) being a particularly challenging target. In this article, we will have an in-depth look at the award-winning SOLIS technique (scanned oblique light-sheet instant-volume sectioning), a new twist on multifocus fluorescence microscopy. Through its unique capabilities, SOLIS offers a new approach on organoid and tissue imaging, providing unprecedented insights into the cellular architecture of these complex artificial samples. By being able to record optically sectioned volumes during single camera exposures, SOLIS demonstrates remarkable advantages over traditional multifocus microscopy, underscoring its potential to transform our understanding of developmental biology, disease mechanisms, and potential therapeutic interventions

A joint Richardson-Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data.

We demonstrate the reconstruction of images obtained by multifocal structured illumination microscopy, MSIM, using a joint Richardson-Lucy, jRL-MSIM, deconvolution algorithm, which is based on an underlying widefield image-formation model. The method is efficient in the suppression of out-of-focus light and greatly improves image contrast and resolution. Furthermore, it is particularly well suited for the processing of noise corrupted data. The principle is verified on simulated as well as experimental data and a comparison of the jRL-MSIM approach with the standard reconstruction procedure, which is based on image scanning microscopy, ISM, is made. Our algorithm is efficient and freely available in a user friendly software package.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1) and by the Medical Research Council (grant MR/K015850/1). FS wishes to acknowledge support from the Studienstiftung des deutschen Volkes and the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG).This was originally published in Methods and Applications in Fluorescence (F Ströhl, CF Kaminski, Methods and Applications in Fluorescence 2015, 3, 014002

A concept for single-shot volumetric fluorescence imaging via orthogonally polarized excitation lattices.

The deconvolution of widefield fluorescence images provides only guesses of spatial frequency information along the optical axis due to the so called missing cone in the optical transfer function. Retaining the single-shot imaging speed of deconvolution microscopy while gaining access to missing cone information is thus highly desirable for microscopy of volumetric samples. Here, we present a concept that superimposes two orthogonally polarized excitation lattices with a phase-shift of p between them. In conjunction with a non-iterative image reconstruction algorithm this permits the restoration of missing cone information. We show how fluorescence anisotropy could be used as a method to encode and decode the patterns simultaneously and develop a rigorous theoretical framework for the method. Through in-silico experiments and imaging of fixed biological cells on a structured illumination microscope that emulates the proposed setup we validate the feasibility of the method

Superresolving the kidney-a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier.

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney's glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world. Graphical abstract

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors.

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council [EP/H018301/1, EP/G037221/1]; Alzheimer Research UK [ARUK-EG2012A-1]; Wellcome Trust [089703/Z/09/Z] and Medical Research Council [MR/K015850/1, MR/K02292X/1]. We thank K. O’Holleran for assistance with the design of the microscope, and L. Shao and R. Heintzmann for useful discussions and suggestions

Arbeitsbericht Nr. 2020-03, September 2020

Fehlende Werte stellen in zahlreichen praktischen Anwendungen vie-mehr den Regelfall als eine Ausnahme dar, erweisen sich aber bei vielen statistischen Verfahren als störend. Die vorliegende Studie untersucht die Auswirkungen von fehlenden Werten auf die Ergebnisse der multiplen linearen Regression. Dazu werden zunächst spezielle Formen von fehlenden Daten und ausgewählte Verfahren zum Umgang mit diesen vorgestellt. Im Rahmen einer Simulationsstudie werden anschließend die Auswirkungen von verschiedenen Ausfallquoten und -mechanismen anhand von sechs empirischen Datensätzen untersucht. Neben einer Analyse verschiedener Einflussgrößen erfolgt ein Vergleich der vorgestellten Verfahren zur Behandlung der fehlenden Werte. Es zeigt sich, dass keines der untersuchten Verfahren allen anderen Verfahren in jeder Hinsicht überlegen ist und die Wahl des „besten“ Verfahrens von der Struktur des Datensatzes und der späteren Verwendung der Regressionsfunktion abhängt. Darüber hinaus konnte festgestellt werden, dass eine Erhöhung der Ausfallquote im Allgemeinen zu einer Verschlechterung der Ergebnisse führt. Die Einflüsse der Objekt- und Merkmalsanzahl hängen von dem jeweiligen Verfahren und den weiteren Eigenschaften des Datensatzes ab und sollten stets zusammen betrachtet werden

Integration von Gewichtsrestriktionen in das DEA-Modell nach Charnes, Cooper und Rhodes: exemplarische Optionen und Auswirkungen

Ziel des Beitrags ist es, die Integration von Gewichtsrestriktionen in das DEA-Basismodell nach Charnes, Cooper und Rhodes (CCR-Modell) zu untersuchen. Für ein fiktives Beispiel werden die Ergebnisse der um verschiedene Gewichtsrestriktionen erweiterten DEA-Modelle präsentiert und diskutiert. In einfachen Sensitivitätsanalysen wird der Einfluss ausgewählter Gewichtsrestriktionen auf die Effizienzwerte näher beleuchtet

Label-free superior contrast with c-band ultra-violet extinction microscopy

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample’s refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup

Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element.

Super-resolution single-molecule localization microscopy, often referred to as PALM/STORM, works by ensuring that fewer than one fluorophore in a diffraction-limited volume is emitting at any one time, allowing the observer to infer that the emitter is located at the center of the point-spread function. This requires careful control over the incident light intensity in order to control the rate at which fluorophores are switched on; if too many fluorophores are activated, their point-spread functions overlap, which impedes efficient localization. If too few are activated, the imaging time is impractically long. There is therefore considerable recent interest in constructing so-called 'top-hat' illumination profiles that provide a uniform illumination over the whole field of view. We present the use of a single commercially-available low-cost refractive beamshaping element that can be retrofitted to almost any existing microscope; the illumination profile created by this element demonstrates a marked improvement in the power efficiency of dSTORM microscopy, as well as a significant reduction in the propensity for reconstruction artifacts, compared to conventional Gaussian illumination